Numerous recent advances in the study of biology have benefited from improved methods of analysis and sequencing of nucleic acids. For example, the Human Genome Project has determined the entire sequence of the human genome, which is hoped to lead to further discoveries in fields ranging from treatment of disease to advances in basic science. While the “human genome” has been sequenced there are still vast amounts of genomic material to analyze, e.g., genetic variation between different individuals and tissues, additional species, etc.
However, along with nucleic acid sequencing is the need for amplification of nucleic acids, thereby allowing small amounts of nucleic acid to be easily detected and sequenced. Many methods such as Polymerase Chain Reaction (PCR) and the like currently exist to amplify nucleic acid. PCR (see, e.g., Saiki et al. 1985, Science 230: 1350) has become a standard molecular biology technique for amplification of nucleic acid molecules. This in vitro method can be a powerful tool for the detection and analysis of small quantities of nucleic acids, however, PCR can have disadvantages in particular applications.
Briefly, the PCR reaction (typically requiring a target nucleic acid molecule, a molar excess of a forward and reverse primers complementary to the target, deoxynbonucleoside triphosphates (dATP, dTTP, dCTP and dGTP) and a polymerase enzyme) is a DNA synthesis reaction that depends on the extension of forward and reverse primers annealed to opposite strands of a double stranded DNA template that has been denatured (melted apart) at high temperature (90° C. to 100° C.). Copies of the original template DNA are generated through repeated melting, annealing and extension steps, carried out at differing temperatures.
Although there have been many improvements and modifications to the original PCR procedure, PCR continues typically to rely on thermocycling of a reaction mixture, with melting, annealing and extension performed at different temperatures. One major disadvantage of thermocycling reactions relates to the long “lag” times during which the temperature of the reaction mixture is increased or decreased to the correct level. These lag times increase considerably the length of time required to perform an amplification reaction. Additionally, the elevated temperatures required with PCR are not ideal for certain applications, especially where nucleic acid molecules are surface bound.
Moreover, as a result of the high temperatures used during PCR, the reaction mixtures are subject to evaporation. Consequently, PCR reactions are carried out in sealed reaction vessels. However, the use of such sealed reaction vessels has further disadvantages since use of a sealed reaction vessel makes it difficult to alter or add further reaction components. For example, as amplification progresses, depletion of dNTPs can become limiting, thereby, lowering the efficiency of the reaction. Repeated high temperature cycling can also lead to a reduction in the efficiency of the polymerase enzyme; the half life of Taq polymerase may be as low as 40 minutes at 94° C. and 5 minutes at 97° C. (see Wu et al. 1991, DNA and Cell Biology 10, 233-238; Landegren U. 1993, Trends Genet 9, 199-204; and Saiki et al. 1988, Science, 239, 487-491). Again, since the reaction vessels are sealed in PCR, addition of fresh dNTPs or Taq is problematic.
To overcome these technical disadvantages there is a continuing need for better, more economical devices and systems for fast reliable nucleic acid amplification where the amplification method does not rely on temperature cycling. The current invention provides these devices (and methods of their use) as well as other benefits which will be apparent upon examination of the current specification, claims, and figures.